Bacillus-derived transglutaminase

ABSTRACT

A method for processing a protein, a non-proteinaceous amino acid polymer, or a non-proteinaceous amino acid polymer, or a peptide or derivatives thereof having a crosslinked structure, which entails contacting glutamine and lysine residues in a protein, a non-proteinaceous amino acid polymer, a peptide or derivatives thereof with a transglutaminase obtained from Bacillus subtilus to form intermolecular or intramolecular, crosslinked ε(δ-Glu)-Lys bonds between or in the molecules of the protein, non-proteinaceous amino acid polymer, peptide or derivatives thereof, wherein the transglutaminase has the physicochemical properties described herein.

This application is a Division of application Ser. No. 08/596,864, filedon Feb. 9, 1996, now U.S. Pat. No. 5,731,183.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to (1) a transglutaminase (hereinafterreferred to as TG) isolated from a Bacilli such as those of Bacillussubtilis, (2) a fraction having transglutaminase activity, and (3) amethod for producing a protein, a non-proteinaceous amino acid polymer,a peptide or derivatives thereof having a crosslinked structure, bycrosslinking the glutamine and lysine residues in the same with the TGor the fraction having TG activity to thereby form intermolecular orintramolecular, crosslinked ε-(γ-Glu)-Lys bonds.

The present invention also relates to (4) a DNA coding for a TG derivedfrom a Bacilli such as Bacillus subtilis, (5) a vector comprising saidDNA coding for said TG, (6) a cell transformed with the vector, and (7)a method for producing a Bacillus-derived transglutaminase by incubatingthe transformant.

The crosslinked polymers produced using the Bacillus-derived TG of thepresent invention can be used in foods such as tofu (soybean curd),pudding, yogurt, cheese, ground fish meat, boiled fish paste, sausageand other fish and meat products and also in cosmetics, etc.

2. Discussion of the Background

TG is an enzyme which catalyzes the transacylation of γ-carboxyamidegroups in the glutamine residues in a peptide chain with the eitherε-amino group in a lysine residue in the peptide chain or water. When aε-amino group is the acyl acceptor, crosslinked ε-(γ-Glu)-Lys bonds(hereinafter referred to as "GL bonds") are formed in or between thepeptide molecules. Where water is the acyl receptor in thetransacylation, the glutamine residue in the peptide chain is subjectedto de-amidation by which the glutamine residue is converted into aglutamic acid residue.

It is known that TG exists in many animal tissues. For example, TGexisting in the liver of guinea pigs has been studied (see Connellan etal., J. Biol. Chem., Vol. 246, pp. 1093-1098, 1971).Microorganism-derived TGs are less well known, only TG derived fromActinomycetes (ray fungi), Bacillus subtilis (see Ramanujam et al.,FASEB J. Vol. 4, A2321) and Myxomycetes (slime molds) (see Klein et al.,J. Bacteriol., Vol. 174, pp. 2599-2605) have been reported. At present,TG produced by ray fungi has been put to practical and industrial use(see Japanese Patent Publication No. 6-65280, Japanese Patent Laid-OpenNo. 1-27471).

Unfortunately, TG derived from animals such as guinea pigs isimpractical for use in industry because it is difficult to obtain alarge amount of such animal-derived TG at low costs. In addition, theanimal-derived TG requires calcium ions, thus limiting its use.

Ray fungus-derived TG also has some drawbacks. Since ray fungi grow moreslowly than ordinary bacteria, they need a long period of time forincubation, resulting in the increase in the costs in producing TG.

Ramanujam et al. of New Mexico State University have reported theexistence of Bacillus subtilis-derived TG. The TG as reported by themhave the following properties:

1) The pH suitable for it is 9.5 or higher.

2) Since its activity is greatly inhibited by a chelating agent (EGTA),it is considered that the TG has the property of requiring metal ions.

3) It is inhibited by Ca²⁺ of 5 mM or more.

4) It is inhibited by dithiothreitol (DTT).

5) It is produced by both vegetative cells and sporulating cells.

It is considered that TG reported by Ramanujam et al. is limited due tothe above-mentioned properties, especially because its operating pH ishigh and it is influenced by metal ions.

Thus, (1) the animal-derived TG is impractical for industrial usebecause it requires calcium increasing production costs, (2) the rayfungus-derived TG is impractical for industrial use because the growthof ray fungi is slow increasing production costs, and (3) Ramanujam etal.'s Bacillus subtilis-derived TG is impractical for industrial use,since it cannot be used in foods since it is inhibited by 5 mM of Ca²⁺.

SUMMARY OF THE INVENTION

Therefore, the object of the present invention is to isolate a novel TGfrom a Bacilli that has previously been used in producing foods, such asBacillus subtilis, and to provide a method for producing crosslinkedpolymers by the use of such TG.

The present inventors have such a TG. The present invention includes aBacillus-derived TG, and a method for producing crosslinked protein,non-proteinaceous amino acid polymer, peptide or derivatives thereofusing the TG, or a fraction comprising the TG. The TG of the presentinvention has the following physicochemical properties:

a) it is active between about pH 7 and about 9,

b) it is active between about 40° C. and about 65° C.,

c) it is stable at about 60° C. or lower,

d) it is independent of Ca²⁺ and has an activity of 50% or more in thepresence of 5 mM of Ca²⁺,

e) it is inhibited by NEM, cystamine and (NH₄)₂ SO₄,

f) it is not inhibited by EDTA, DTT and 2-ME,

g) it has a molecular weight of (i) from about 18,000 to about 22,000 asmeasured by gel permeation and (ii) from about 28,000 to about 30,000 asmeasured by SDS-PAGE, and

h) it catalyzes the transacylation of the γ-carboxyamide group inglutamine residue(s) in a peptide chain.

The present invention further includes a DNA coding for the above TG, avector comprising such DNA, a cell transformed with the vector, and amethod for producing a Bacillus-derived transglutaminase by incubatingthe transformant.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete appreciation of the invention and many of the attendantadvantages thereof will be readily obtained as the same becomes betterunderstood by reference to the following detailed description whenconsidered in connection with the accompanying drawings, wherein:

FIG. 1 shows the results of SDS-PAGE of pure TG-1.

FIG. 2 shows the pH curve relative to the activity of TG-1.

FIG. 3 shows the temperature curve relative to the activity of TG-1.

FIG. 4 shows the temperature stability of TG-1.

FIG. 5 shows a-casein as crosslinked with TG-1.

FIG. 6 shows the relationship between the growth of TG-producing cellsand the activity of TG-2 produced.

FIG. 7 shows the pH curve relative to the activity of TG-2.

FIG. 8 shows the temperature curve relative to the activity of TG-2.

FIG. 9 shows the temperature stability of TG-2.

FIG. 10 shows the influence of inhibitors on TG-2.

FIG. 11 shows the influence of DTT on TG-2.

FIG. 12 shows the influence of EDTA on TG-2.

FIG. 13 shows the influence of Ca²⁺ ions on TG-2.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Protein, non-proteinaceous amino acid polymer, peptide and derivativesthereof having crosslinked structures can be formed by the action of theTG or the TG activity-having fraction are referred to herein ascrosslinked polymers.

The TG of the present invention can be isolated from sporogenicbacteria, such as Bacilli, typically those of Bacillus subtilis.Preferably the bacteria are at the sporulation stage. Two preferredstrains include Bacillus subtilis AJ12866 and Bacillus subtilis AJ1307.

Bacillus subtilis AJ12866 was deposited on Feb. 2, 1995 in the NationalInstitute of Bioscience and Human-Technology, the Agency of IndustrialScience and Technology, the Ministry of International Trade and Industryof Japan (hereinafter referred to as "NIBH") under accession number FERMP-14750. Bacillus subtilis AJ12866 was then transferred to theinternational depository on Dec. 4, 1995, under the provisions of theBudapest Treaty, under international deposit number FERM BP-5325.

Bacillus subtilis AJ1307 was deposited on Aug. 22, 1995 in NIBH, underaccession number FERM P-15123. Bacillus subtilis AJ1307 was thentransferred to the international depository on Jan. 18, 1996, under theprovisions of the Budapest Treaty, under international deposit numberFERM BP-5367.

The TGs of the present invention broadly exist in bacilli having spores.Namely, the TG also exists in Bacillus licheniformis, Bacillusmegaterium, Bacillus stearothermophilus, Bacillus brevis, Bacillussphaericus, Bacillus polymyxa, Bacillus alcalophilus, etc.

Bacilli can be incubated using techniques known in the art by liquidcultivation or solid cultivation. In particular, deep aerating andstirring cultivation is industrially advantageous.

Suitable nutrient sources in nutrient media for the bacilli includeordinary carbon sources, nitrogen sources, inorganic salts and otherminor nutrients that are generally used in incubation of microorganisms.All nutrient sources known for use with Bacilli can be employed.

During aeration, aerobic conditions are employed. Bacilli can beincubated at any temperature at which they can grow and produce TG.Typically, the incubation temperature is from 10 to 50° C., preferablyfrom 30 to 40° C. Bacilli which are thermophilic bacteria can beincubated at temperatures higher than the above-mentioned range.

The incubation time will vary, depending on the incubation time andother incubation conditions. Preferably, bacilli are incubated for along period of time such that they produce the largest amount of TG. Ingeneral, Bacillus are incubated for from 5 hours to 7 days or so,preferably for from 10 hours to 3 days or so.

The stage at which Bacilli exhibit their TG activity according to thepresent invention is limited to only the sporulation stage. This is themost significant point in which the TG of the present invention isbasically different from the Bacillus subtilis-derived TG as reported bythe group of the New Mexico State University.

After the incubated cells of Bacilli begin to form spores, their TGactivity begins to increase. Then, the TG activity becomes the largestat the sporulation stages IV to VI or so, and thereafter it decreases.TG activity can be slightly detected in the culture but can be detectedmuch more in the grown cells.

The thus-grown cells are disrupted or lysed under low-temperatureconditions. The thus-processed cells are centrifuged at 20000×g for 10minutes. Then, the supernatant fraction and the precipitate fraction areseparated from each other, and the TG activity of each fraction isdetected. In that manner, it is verified that the precipitate fractionthat contains the spores has TG activity. Thus, TG exists on thesurfaces of the spores.

To purify the Bacillus-derived TG, the culture comprising the growncells of Bacilli may be directly processed to obtain a purified TG, butit is advantageous that the sporangia of the grown cells are firstdisrupted or lysed and then the resulting spores are processed to obtaina purified TG.

By disrupting or lysing the sporangia to be obtained by incubatingBacilli, it is possible to obtain the spores of Bacilli. After thedisrupting or lysing treatment, the fraction having TG activity iscollected in the insoluble fraction containing the spores. Therefore, itis also possible to concentrate the insoluble fraction to obtain theintended enzyme preparation.

In order to recover the TG activity that has been collected in theinsoluble fraction in a soluble fraction (that is, in order tosolubilize the TG activity), the following operations are needed.

First, a surfactant such as TRITON X-100 (a polyethylene ethersurfactant), alkylglucoside or the like can be added to the insolublefraction. Second, the spore-containing fraction can be treated with abasic buffer (for example, 20 mM sodium bicarbonate buffer, pH 10).Third, the spore-containing fraction can be suspended in a buffer andheated. In all of the above, the TG activity is recovered in theresulting soluble fraction. For example, by heating the suspension at10° C. or higher, the TG activity can be recovered in the solublefraction.

The solubilized TG can be utilized as a gelling agent. By employing anyordinary methods of, for example, gel permeation, ion-exchangechromatography, etc., which can be used for purifying enzyme, thesolubilized TG can be further purified. As a result, TG having a higherrelative activity can be obtained. The thus-purified TG can be a gellingagent having a higher relative TG activity.

The measurement of the TG activity shall be conducted in the mannermentioned below. ¹⁴ C-labeled putrescine and dimethylcasein are used asthe substrates, and a sample containing TG is applied to these to makethem reacted with each other. The putrescine-bonded dimethylcasein isprecipitated with 10% TCA, and the resulting precipitate is adsorbedonto filter paper. Since the radioactivity existing in the filter paperis proportional to the TG activity in the sample, it is possible toquantitatively determine the TG activity in the sample. Theradioactivity can be measured with a liquid scintillation counter.

The Bacillus subtilis AJ1307-derived TG is hereinafter referred to asTG-1, while the Bacillus subtilis AJ12866-derived TG is as TG-2. On thebasis of the data of TG-1 and TG-2, the physicochemical properties of TGof the present invention are mentioned below.

Suitable pH Range:

The pH suitable for TG of the present invention falls between about 7and about 9 or so.

In order to determine the pH range within which the TG is active,various enzymatic reactions with the TG were carried out at 37° C. for30 minutes.

Suitable Temperature Range:

The temperature range within which the TG is active is between about 40°C. and about 65° C. or so.

In order to determine the temperature range within which the TG issuitably active, various enzymatic reactions with the TG were carriedout at pH of 7.5 for 30 minutes.

Temperature Stability:

The TG was stable at about 60° C. or lower.

The TG was subjected to high-temperature treatment at pH of 7.5 for 10minutes, whereupon the temperature stability of the TG was checked. Evenwhen the TG was subjected to high-temperature treatment at 60° C., itstill maintained about 80% of the TG activity.

Influence of Inhibitors:

The Bacillus-derived TG of the present invention is greatly inhibited byNEM (N-ethylmaleimide) and cystamine. In addition, it is also greatlyinhibited by (NH₄) ₂ SO₄ (ammonium sulfate).

Influence of DTT and EDTA:

The activity of the Bacillus-derived TG of the present inventionincreases in the presence of DTT (dithiothreitol). However, the TGactivity was not influenced by the presence of EDTA(ethylenediamine-tetraacetic acid).

Influence of Ca²⁺ :

The TG of the present invention does not have the property of requiringCa²⁺ ions. Namely, it is a Ca²⁺ -independent enzyme. The TG stillmaintains 50% or more of its activity in the presence of 5 mM of Ca²⁺.

Molecular Weight:

The TG has a molecular weight of (a) from about 18,000 to about 22,000(as measured by gel permeation) and (b) from about 28,000 to about30,000 (as measured by SDS-PAGE).

Activity:

The TG catalyzes the transacylation of a substrate, γ-carboxyamide groupin the glutamine residue existing in a peptide chain. When the ε-aminogroup in a lysine residue in the peptide chain is the acyl receptor,intramolecular or intermolecular, crosslinked ε-(γ-Glu)-Lys bonds areformed in or between the peptide molecules. When water is an acylreceptor in the transacylation, the glutamine residue in the peptidechain is subjected to de-amidation by which the glutamine residue isconverted into a glutamic acid residue.

As mentioned hereinabove, the properties of TG of the present inventionthat is derived from Bacillus subtilis AJ12866 and Bacillus subtilisAJ1307 were obviously different from those of the Bacillussubtilis-derived TG as reported by the group of the New Mexico StateUniversity.

The TG of the present invention can be used to produce crosslinkedpolymers. The TG can be used in a variety of forms including (1) aconcentrate of the insoluble, spore-containing fraction that is obtainedby disrupting or lysing the sporangia of bacilli incubated, (2) a TGactivity-having fraction that is obtained by solubilizing the insolublefraction in various manners, and (3) a purified TG having a highrelative activity. Alternatively, any and every other fraction havingBacillus-derived TG activity can be used.

It is also possible to use a Bacillus-derived TG that is obtained byincubating cells transformed with a vector comprising DNA coding for aBacillus-derived TG. Such a vector can be obtained using methods knownin the art such as those mentioned below.

Suitable substrates for the TG or TG-active fraction include one or moreof proteins, non-proteinaceous amino acid polymers, peptides andderivatives thereof which contain at least one glutamine residue and atleast one lysine residue. The origins and the properties of the proteinsare not specifically defined. Suitable proteins include casein, gelatin,and soybean protein. In addition, proteins denatured partly byproteases, etc. can also be employable.

Suitable non-proteinaceous amino acid polymers include amino acidpolymers to be produced by chemical synthesis, such as polylysine, etc.The peptides may be those as obtained by chemical synthesis or those asobtained by decomposing natural proteins with acids, alkalis, proteases,etc. Suitable derivatives of such substances include glycoproteins,chemically-modified proteins, etc. Any other protein or fragment thereofcan also be used as the substrate for the TG or TG-active fraction,provided that they have lysine and glutamine residues.

The TG or TG-active fraction of the present invention can be added toand reacted with a solution or slurry containing a protein or the likeat a substrate concentration of 0.1% or more, whereby a crosslinkedpolymer product is obtained. The crosslinked polymers to be obtainedaccording to the present invention may be classified into a group ofgelled products, a group of highly-viscous products and a group ofmerely-polymerized products, depending on the degree of crosslinking inthem. The present invention encompasses all such crosslinked polymers.

In general, the pH of the reaction solution is from about 4 to about 10,the reaction temperature is from about 5° C. to about 80° C., and thereaction time is from about 10 seconds to about 24 hours. As a result ofthe reaction, crosslinked polymers (gelled products, highly-viscousproducts, etc.) are obtained.

Many examples of producing useful proteins such as enzymes,physiologically-active substances, etc. by recombinant DNA technologyare known. One advantage of recombinant DNA technology is that it ispossible to produce large amounts of useful proteins that exist onlyslightly in the natural world. To produce the Bacillus-derived TG of thepresent invention according to such recombinant DNA technology, a DNAcoding for the Bacillus-derived TG is needed. The DNA is linked to avector DNA to construct the intended recombinant DNA.

DNA encoding the Bacillus-derived TG of the present invention can beobtained as mentioned below. First, the amino acid sequence of thepurified TG is determined. It is possible to determine the intendedamino acid sequence according to the Edman method (see Edman, P., ActaChem. Scand., 4, 227 (1950)). In addition, it is also possible todetermine the amino acid sequence by the use of a sequencer produced byApplied Biosystems Co.

The amino acid sequence of from the N-terminal to the 35th residue ofthe Bacillus-derived TG of the present invention is shown in theSequence Listing as SEQ ID NO:1.

On the basis of the thus-clarified amino acid sequence, the basesequence of the DNA that codes for this can be deduced. To deduce thebase sequence of the DNA, employed are a universal codon or a codon thatis most frequently used in the genes of Bacilli.

On the basis of the thus-deduced base sequence, DNA molecules havingfrom 30 to 50 base pairs or so are synthesized. The method forsynthesizing such DNA molecules is described in Tetrahedron Letters, 22,1859 (1981). The DNA molecules can also be synthesized by the use of asynthesizer produced by Applied Biosystems Co. The DNA molecules can beused as probes, when the whole length DNA that codes for theBacillus-derived TG is isolated from the Bacillus chromosome genelibrary. In addition, these can also be used as primers, when the DNAthat codes for the Bacillus-derived TG is amplified by PCR. However,since the DNA as amplified by PCR does not include the whole length DNAthat codes for the Bacillus-derived TG, the DNA as amplified by PCR isused as the probe and the whole length DNA that codes for theBacillus-derived TG is isolated from the Bacillus chromosome genelibrary.

Suitable operations for PCR are described by White et al., in TrendsGenet., 5, 185 (1989). The method for preparing the chromosome DNA ofbacilli is described in Molecular Biological Methods for Bacillus, JohnWiley & Sons, Ltd. (1990), etc. The method for constructing thechromosome gene library of Bacilli, etc., is described in MolecularBiological Methods for Bacillus, John Wiley & Sons, Ltd. (1990), etc.The method of isolating the intended DNA molecule from the gene libraryby using DNA molecules as probes is described in Molecular Cloning, 2ndEdition, Cold Spring Harbor Press (1989), etc.

To determine the base sequence of the DNA that codes for theBacillus-derived TG and that has been isolated according to the mannermentioned above, referred to is the method as described in "A PracticalGuide to Molecular Cloning", John Wiley & Sons, Inc. (1985). For thebase sequencing, also employable is a DNA sequencer produced by AppliedBiosystems Co.

One DNA that codes for the Bacillus-derived TG of the invention is shownas SEQ ID NO:2 in the Sequence List. This DNA was isolated from thechromosomal DNA of Bacillus subtilis AJ1307. The DNA that codes for theBacillus-derived TG is not limited to that shown as SEQ ID NO:2 in theSequence List. The DNA encoding the TG of the present invention can haveany base sequence, in accordance with the species and the strain of theBacillus from which it is derived.

It is possible to artificially mutate the DNA that codes for the TGisolated from the chromosomal DNA of a Bacillus to modify the basesequence of the DNA. One popular method for such artificial mutation isa site-specific mutation method such as that described in Method inEnzymol., 154 (1987). Any artificially-mutated DNAs, if coding for theBacillus-derived TG, are within the scope of the DNA that codes for theBacillus-derived TG of the present invention.

Host cells are transformed with the recombinant DNA. The transformantcells that have been transformed to be able to produce theBacillus-derived TG are incubated in a medium to thereby make the cellsproduce and accumulate the Bacillus-derived TG in the medium or in thecells, and thereafter the TG is collected.

Cells of Escherichia coli AJ13172 that have the recombinant DNA asconstructed by linking the DNA coding for the Bacillus subtilisAJ1307-derived TG to a vector DNA were deposited with internationaldepository NIBH on Dec. 20, 1995 under the provisions of the BudapestTreaty, under international deposit number of FERM BP-5346.

It is possible to produce a large amount of the Bacillus-derived TG bylinking a vector DNA to the DNA that codes for the Bacillus-derived TGto construct a recombinant DNA, then transforming cells with therecombinant DNA to give transformant cells, incubating the transformantcells in a medium to make the cells produce and accumulate theBacillus-derived TG in the medium and/or in the cells, and collectingthe TG.

In many cases where a large amount of protein is desired to be producedby recombinant DNA technology, the protein produced is oftenintramolecularly associated to give its inclusion body in thetransformant that is producing the protein. The advantages of theexpression method of producing protein are that the intended protein canbe protected from being digested by the protease existing in the cellsused and that the intended protein can be easily purified by disruptingthe cells followed by centrifuging the resulting cell debris.

The protein inclusion body thus obtained is solubilized with aprotein-denaturating agent, then the activity of the thus-solubilizedprotein is regenerated essentially by removing the denaturating agent,and thereafter the soluble protein is converted into a correctly-folded,physiologically-active protein. There are known many examples relatingto the process, and one example is to regenerate the activity of humaninterleukin-2 (see Japanese Patent Laid-Open No. 61-257931).

To obtain the activated protein from the protein inclusion body, aseries of operations for solubilization, regeneration of the activity,etc. is necessary, and such operations are more complicated than thosefor directly producing the activated protein. However, when a largeamount of protein is produced in cells and when the protein thusproduced has an influence on the growth of the cells, the protein can beaccumulated in the cells in the form of such an inactive proteininclusion body whereby the influence of the protein can be retarded.

As the method for producing a large amount of the intended proteininclusion body, there are known a method of expressing the intendedprotein by itself under the control of a strong promoter and a method ofexpressing the intended protein as a fused protein with a differentprotein that is known to be expressed in large quantities.

In addition, it is also effective to previously insert some restrictionprotease recognition site into the fused protein at a suitable position,via which the intended protein can be cut out of the fused protein afterits expression.

The host cells to be transformed for the production of a large amount ofprotein by recombinant DNA technology include cells of bacteria, cellsof ray fungi, cells of yeasts, mold cells, vegetative cells, animalcells, etc. In general, cells of a colon Bacillus, Escherichia coli arepreferably employed. This is because there are many prior art techniquesfor producing a large amount of protein by the use of cells ofEscherichia coli.

As the promoter for expressing a DNA that codes for the Bacillus-derivedTG, usable are promoters which are generally used for making Escherichiacoil produce foreign proteins. For example, usable are strong promoterssuch as T7 promoter, trp promoter, lac promoter, tac promoter, PLpromoter, etc.

In order to make the host cells produce the Bacillus-derived TG as afused protein inclusion body, a gene that codes for another protein,preferably a hydrophilic peptide shall be linked to the upstream ordownstream site of the Bacillus-derived TG gene in each host cell toconstruct a fused protein gene therein. The gene that codes for anotherprotein may be any one that has the ability to increase the amount ofthe intended fused protein to be accumulated in the host cells and toenhance the solubility of the fused protein after the denaturation andregeneration of the fused protein. As candidates for the gene, forexample, mentioned are T7 gene 10, β-galactosidase gene, dehydrofolicacid reductase gene, interferon γ-gene, interleukin-2 gene, prochymosingene, etc.

When any of these genes is linked to the gene that codes for theBacillus-derived TG, the codon reading frames for the two genes shall bethe same. Either the genes are linked to each other at suitablerestriction endonuclease sites or any synthetic DNA having anappropriate sequence is utilized.

In order to increase the amount of the Bacillus-derived TG to beproduced by the host cells, it is preferable to link a terminator thathas a transcription-terminating sequence to the downstream site of thefused protein gene. The terminator includes, for example, T7 terminator,fd phage terminator, T4 terminator, tetracycline-resistant geneterminator, E. coil trpA gene terminator, etc.

As the vector via which the gene that codes for the Bacillus-derived TGor codes for a fused protein composed of the Bacillus-derived TG andanother protein is introduced into Escherichia coli, preferred is aso-called multicopying vector. The vector includes, for example, pUCplasmids, pBR322 plasmids and their derivatives. In order to favorablyconduct the screening of the resulting transformants, it is desirablethat the vector contain a marker such as an ampicillin-resistant gene,etc. As such plasmids, various expression vectors having a strongpromoter are commercially available (for example, pUC plasmids producedby Takara Shuzo Co., pPROK plasmids produced by Clonetec Co., pKK233-2produced by Clonetec Co., etc.).

A DNA fragment comprising a promoter, a gene that codes for theBacillus-derived TG or codes for a fused protein composed of theBacillus-derived TG and another protein, and a terminator as linked inthat order is linked to a vector DNA to construct a recombinant DNA.

Cells of Escherichia coil are transformed with the recombinant DNA, andthe resulting transformant cells are incubated to make them express andproduce the Bacillus-derived TG or the fused protein composed of theBacillus-derived TG and another protein.

As the hosts to be transformed, any strains which are generally used forexpression of foreign genes can be employed. Especially preferred areEscherichia coli JM109(DE3) strain and JM109 strain. The method fortransformation and the method for screening the resulting transformantsare described in "Molecular Cloning", 2nd Edition, Cold Spring HarborPress (1989), etc.

Where a fused protein is expressed, it is possible to modify it suchthat TG can be cut out of the fused protein using a restriction proteasewhich has, as the recognition sequence, a sequence not existing in TG,such as a blood coagulation factor Xa, kallikrein or the like.

As the production media, usable are any ordinary media which aregenerally used for incubating cells of Escherichia coli, such asM9-Casamino medium, LB medium, etc. The incubation conditions and theproduction-inducing conditions shall be suitably selected in accordancewith the vector marker, the promoter, the type of the host cells used,etc.

To recover and collect the Bacillus-derived TG or the fused proteincomposed of the Bacillus-derived TG and another protein, for example,employable are various methods such as those mentioned below. Where theTG or the fused protein has been solubilized in the incubated cells, thecells are collected and then disrupted or lysed and the thus-obtained,cell debris-containing liquid can be used as a crude enzyme liquid. Ifdesired, the liquid is further subjected to ordinary precipitation,filtration, column chromatography or the like to thereby purify the TGor the fused protein before use.

If desired, a method of purifying the TG or the fused protein by the useof an antibody thereto can also be employed.

Where a protein inclusion body is formed, this is solubilized with aprotein-denaturating agent. In this case, the protein inclusion body canbe solubilized along with the cells containing it. However, inconsideration of the subsequent operations for purification, it ispreferable to isolate the inclusion body prior to the solubilizationthereof. The isolation of the inclusion body from the cells can beconducted by any known methods. For example, the cells are disrupted andthen subjected to centrifugation or the like through which the inclusionbody is recovered.

As the protein-denaturating agent with which the protein inclusion bodyis solubilized, usable are guanidine hydrochloride (for example, at aconcentration of 6 M and at a pH of from 5 to 8), urea (for example, ata concentration of 8 M), etc.

The protein-denaturating agent used is removed by dialysis or the like,and the active protein is regenerated. The dialyzing solution to be usedfor the dialysis may be a tris-HCl buffer, a phosphate buffer or thelike. Its concentration may be from 20 mM to 0.5 M and its pH may befrom 5 to 8.

It is desirable that the protein concentration during the regenerationstep is restricted to be not higher than 500 μg/ml or so. In order toprevent the thus-regenerated Bacillus-derived TG from beingself-crosslinked, it is desirable that the dialysis temperature is nothigher than 5° C. To remove the protein-denaturating agent, alsoemployable are a dilution method, an ultrafiltration method, etc., inaddition to the above-mentioned dialysis method. Any of these methodsare employable to attain the intended regeneration of the activity ofthe protein.

Where the DNA having the sequence of SEQ ID NO:2 in the Sequence List isused as the DNA that codes for the Bacillus-derived TG, theBacillus-derived TG having the amino acid sequence shown under the basesequence of SEQ ID NO:2 is obtained. The open reading frame in the DNAranges from the 118th adenosine residue to the 8692nd cytosine residue.

According to the present invention, it is possible to obtain a novel TGwhich has heretofore been unknown from microorganisms, bacilli which areusable in the food industry, such as Bacillus subtilis, etc.

The novel TG of the present invention is advantageous in that (1) itdoes not require calcium, being different from any other animal-derivedTGs, and therefore its use is not limited and it can be produced at lowcost and that (2) the bacilli which produce the TG of the presentinvention grow faster than ray fungi and therefore the Bacillus-derivedTG can be produced at lower costs than the ray fungus-derived TG.

The TG of the present invention is different from the TG as reported bythe group of the New Mexico State University, in that (1) the activityof the former is not inhibited by Ca²⁺ of 5 mM or more, (2) the pH rangesuitable for the former falls from neutral to weak alkaline, (3) theformer is not inhibited by DTT, (4) the former is not inhibited bychelating agents such as EGTA, etc., and (5) the former is produced byBacilli only at the stage of their sporulation.

Since the TG of the present invention can be used for producingcrosslinked polymer substances, it can be applied to the industrialproduction of various foods.

In addition, since the TG of the present invention is derived frombacilli which are practically used in producing foods, its practicalvalue in the food industry is high.

EXAMPLES

Having generally described this invention, a further understanding canbe obtained by reference to certain specific examples which are providedherein for purposes of illustration only and are not intended to belimiting unless otherwise specified.

Example 1 Production and purification of TG

Cells of Bacillus subtilis AJ1307 were incubated to make them have asufficient TG activity. Using Schaeffer's medium, the incubation wasconducted always at 37° C. by liquid shaking cultivation or liquidaerating and stirring cultivation. The Schaeffer's medium used had acomposition comprising 8 g/liter of Bacto-nutrient broth, 1 g/liter ofKCl, 0.12 g/liter of MgSO₄.7H20, 1 mM of CaCl₂, 10 μM of MnCl₂ and 1 μMof FeSO₄ and had pH of 7.0.

First, cells of AJ1307 strain were incubated in 20 ml of one medium for24 hours for seed cultivation. 5 ml of the culture comprising the seedcells were again incubated in a series of three batch media of 100 mleach. After the cells reached the latter stage of logarithmic growthphase in each medium, each culture was transferred to 900 ml of anothermedium. These three batch media were connected in series, where theincubation of the cells was continued. After the cells again reached thelatter stage of logarithmic growth phase in each medium, three liters inall of the cultures were transferred to 27 liters of another medium andwere further incubated therein while aerating at 1/4 vvm and stirring at350 rpm.

Again, after the cells reached the latter stage of logarithmic growthphase, 30 liters of the culture was transferred to 270 liters of anothermedium and was further incubated therein while aerating at 1/20 vvm andstirring at 200 rpm. This is the final incubation. After the cellsreached the stage of stationary phase, the incubation was furthercontinued for 6 hours. After this, the final incubation was finished.The final culture was rapidly cooled to 20° C. or lower, using coldwater. Then, this was centrifuged, using a continuous centrifuger, tocollect the cells. The thus-obtained cells were used as the startingmaterial, from which TG was purified in the manner mentioned below.

The TG activity of the enzyme liquids obtained below was determinedaccording to an enzymatic activity measuring method, which is asfollows. 50 μl of a reagent liquid (100 mM Tris-HCl, pH 7.5, 6.3 mg/mldimethylcasein, 10 nM ₁₄ C-putrescine, 1.2 μci) containing 10 μl of anenzyme liquid to be measured was reacted at 37° C. for 30 minutes, andthen 40 μl of the thus-reacted liquid was adsorbed onto filter paper andfixed thereon with 10% TCA. Next, this was washed three times with a 5%TCA solution, and its radioactivity was counted using a liquidscintillation counter. The thus-counted value was referred to as the TGactivity of the enzyme liquid.

1. Washing of cells:

The cells as collected after the incubation were suspended in 50 mMTris-HCl (pH 7.5) and centrifuged for 30 minutes at 20,000×g to againcollect the cells in the precipitated fraction. The operations for suchsuspension and centrifugation are to wash the cells. Such washing of thecells was repeated for a total of two times.

2. Lysis of cells:

To one part by weight of the thus-washed wet cells, were added 9 partsby weight of Buffer 1 (100 mM Tris-HCl (pH 7.5), 0.5 mg/ml lysozyme, 20μg/ml DNase I, 1 mM EDTA, 2 mM phenylmethanesulfonyl fluoride (PMSF))that had been cooled with ice, and thus the cells were suspended in thebuffer. The resulting suspension was stirred on ice for 1 to 3 hours,whereby the cells were lysed.

3. Preparation of spores:

After the lysis, the resulting solution was centrifuged for 30 minutesat 4° C. and at 20,000×g. The TG activity of each of the centrifugedsupernatant and the suspension as prepared by suspending the centrifugedprecipitate in Buffer 2 (100 mM Tris-HCl (pH 7.5), 1 mM EDTA) wasmeasured. As a result, the TG activity was detected in the precipitatesuspension. The precipitate suspension was observed with a microscope,and the result showed that the suspension contained the bacterial sporesand the lysed cell debris residue.

The precipitate suspension whose TG activity had been detected wasstirred for 30 minutes while cooling with ice. Next, this was againcentrifuged for 30 minutes at 20,000×g, and the TG activity of each ofthe centrifuged supernatant and the suspension as prepared by suspendingthe centrifuged precipitate in Buffer 2 was measured. As a result, theTG activity was detected in the precipitate suspension.

The operations for such suspension, stirring and centrifugation usingBuffer 2 are to wash the spores. Such washing of the spores was repeatedfor a total of four times. During the operations, the TG activity wasalways detected in the precipitate fractions. After the spores were thuswashed for a total of four times, the last suspension of the precipitatefraction whose TG activity had been detected was observed with amicroscope, and the result showed that the suspension contained almostno cell debris residue but contained only the bacterial spores. Themicroscopic observation further revealed the absence of any germinatedspores.

4. Solubilization of TG:

After having been washed, the spores were centrifuged and collected inthe precipitate fraction, and these were suspended in Buffer 3 (0.1 Msodium carbonate, 1 mM EDTA, 50 mM dithiothreitol, pH 10.0) that hadbeen previously heated at 37° C. The pH of the resulting suspension wasadjusted to 10.0, and then the suspension was stirred for 30 minutes at37° C. Next, this was centrifuged for 30 minutes at 20,000×g, and the TGactivity of each of the centrifuged supernatant and the suspension asprepared by suspending the centrifuged precipitate in Buffer 3 wasmeasured.

As a result, the TG activity was detected in the centrifugedsupernatant. The result verifies the solubilization of TG. TheTG-containing solution is referred to as a crude TG solution.

5. Precipitation and removal of concomitant Proteins under acidicconditions:

The crude TG solution was filtered through filter paper, and acetic acidwas added to the resulting filtrate to thereby make the filtrate have pHof 5.8. Then, the filtrate was stirred at 5° C. for 1 hour. As a resultof this operation, a white precipitate was formed, which was consideredto be an isoelectric protein precipitate. Next, this was centrifuged at20,000×g for 30 minutes, whereby the precipitate was separated from thecentrifuged supernatant. The precipitate was dissolved in Buffer 3. TheTG activity of each of the centrifuged supernatant and the solution ofthe precipitate was measured. As a result, the TG activity was detectedin the centrifuged supernatant.

6. Precipitation of TG with ammonium sulfate:

To the centrifuged supernatant whose TG activity had been detected, wasadded 1/20 by volume, relative to the supernatant, of 1 M Tris-HCl (pH7.5). Next, ammonium sulfate was added and dissolved therein at a finalconcentration of 50% saturation. The pH of the resulting solution wasadjusted to 7.5 by adding sodium hydroxide thereto. Next, this wasstirred on ice for 2 hours and then centrifuged at 20,000×g for 30minutes. The thus-centrifuged supernatant was dialyzed against Buffer 4(25 mM Tris-HCl (pH 7.5), 5 mM sodium azide), while the centrifugedprecipitate was dissolved in Buffer 4 and then dialyzed against Buffer4. Through the dialysis, ammonium sulfate was completely removed, andthe TG activity of each of the supernatant fraction and the precipitatefraction was measured. As a result, the TG activity was detected in thefraction of the centrifuged precipitate or, that is, the fraction of theprecipitate as formed in the presence of 50%-saturated ammonium sulfate.

7. Hydrophobic chromatography:

The solution whose TG activity had been detected was dialyzed againstBuffer 5 (50 mM Tris-HCl, 0.75 M magnesium sulfate, 0.02% (w/v) sodiumazide, pH 9.0). The resulting dialysate was centrifuged at 20,000×g for30 minutes to isolate the supernatant. The thus-obtained supernatant wasapplied to a hydrophobic chromatography column, PHENYL SEPHAROSE HP(produced by Pharmacia Co.) that had been equilibrated with the buffer.By this operation, the TG was adsorbed onto the gel.

The protein not adsorbed onto the gel (non-adsorbed protein) was washedaway, using Buffer 5. Next, the adsorbed protein was eluted, using thebuffer containing ethylene glycol as the eluent. In the elution, themagnesium sulfate concentration and the ethylene glycol concentration inthe buffer were varied linearly. Namely, the elution was conducted insuch a way that the magnesium sulfate concentration in the buffer usedwas varied linearly from 0.75 M to 0 M while the ethylene glycolconcentration therein was varied also linearly from 0% (v/v) to 10%(v/v).

The TG activity of each eluate fraction obtained by the elution wasmeasured with the result that the TG activity was observed in the eluateas eluted at the magnesium sulfate concentration of approximately from150 to 200 mM and at the ethylene glycol concentration of approximatelyfrom 7 to 8% (v/v).

8. Gel permeation:

The fraction having TG activity was concentrated, using anultrafiltration device (CENTRIPREP, produced by Amicon Co.), anddialyzed against Buffer 6 (25 mM Tris-HCl, 150 mM NaCl, 1% (v/v)ethylene glycol, 0.02% (2/v) sodium azide, pH 8.0). The resultingdialysate was centrifuged at 20,000×g for 10 minutes to collect theresulting supernatant. The thus-obtained supernatant was applied to agel permeation column, SEPHARACRYL S200HR (produced by Pharmacia Co.)that had been equilibrated with Buffer 6. The TG activity of eachfraction as eluted was measured. As a result, the TG activity wasdetected in the fraction whose molecular weight was estimated to be fromabout 18,000 to about 22,000 or so.

9. Anion-exchange chromatography:

The TG fraction thus obtained was concentrated through anultrafiltration membrane and dialyzed against Buffer 7 (25 mMpiperazine, 1% (v/v) ethylene glycol, 0.02% sodium azide, pH 10.5). Theresulting dialysate was centrifuged at 20,000×g for 10 minutes tocollect the supernatant. The thus-obtained supernatant was applied to ananion-exchange chromatography column, MONO-Q (produced by Pharmacia Co.)that had been equilibrated with Buffer 7. By this operation, the TG wasadsorbed onto the gel.

The non-adsorbed protein was washed away, using Buffer 7. Next, theadsorbed protein was eluted, using the buffer containing NaCl as theeluent. The elution was conducted in such a way that the NaClconcentration in the buffer used was varied linearly from 0 mM to 500mM. The TG activity of each eluate fraction obtained by the elution wasmeasured with the result that the TG activity was observed in the eluateas eluted at the NaCl concentration of approximately from 50 mM to 150mM.

The thus-obtained active fraction was subjected to SDS-PAGE and stainedwith Coomassie Brilliant Blue. From this, it was confirmed that the TGwas purified to show one single band, and it was estimated that themolecular weight of the TG would be from about 28,000 to about 30,000(see FIG. 1).

The increase in the relative activity of the purified fraction wasdetermined. Concretely, the TG activity of the above-mentioned crude TGsolution and that of the purified active fraction were measured. Theresults showed that the relative TG activity per the unit protein weightof the purified fraction increased to about 600 times of that of thecrude solution due to the series of the purification operations. In themethod of measuring the activity as employed herein, the relative TGactivity of the purified fraction was estimated to be about 2.5×104dpm/mg/30 min (37° C., pH 7.5).

10. Determination of the amino acid sequence of TG around itsN-terminal:

The amino acid sequence around the N-terminal of the TG as purified inthe manner mentioned above was determined as follows.

The purified TG fraction of about 10 μg, in terms of the proteintherein, was subjected to polyacrylamide gel electrophoresis in thepresence of SDS, and the TG in the gel was transcribed onto a membranefilter. From this, the amino acid sequence of the TG starting from itsN-terminal was analyzed by the use of a protein sequencer.

Concretely, according to a semi-dry system (see Protein structureAnalysis, written by H. Hirano, published by Tokyo Kagaku Dojin) usingMILLIBLOT (as produced by Millipore Co.), the intended enzyme wastranscribed onto a polyvinylidene fluoride (PVDF) membrane from the gelobtained by the electrophoresis. Next, the N-terminal amino acidsequence of the intended enzyme thus transcribed on the PVDF membranewas sequenced, using a protein sequencer (Model 476 A, produced by ABICo.).

Thus, the amino acid sequence of the TG comprised of 35 residues fromits N-terminal was determined. The amino acid sequence around theN-terminal of the transglutaminase thus sequenced is shown as SEQ IDNO:1 in Sequence List.

Example 2 Determination of pH and temperature ranges suitable for TG:

The pH-dependent variation in the enzymatic activity of the TG wasmeasured according to the method mentioned below, from which the pHrange suitable for the TG was determined.

As the buffers for the enzymatic reaction, used were sodium formatebuffers (pH: 2.0, 3.0, 3.5, 4.0), sodium acetate buffers (pH: 4.5, 5.0,5.5, 6.0), Tris-HCl buffers (pH: 7.0, 7.5, 8.0, 8.5, 9.0) and sodiumcarbonate buffers (pH: 9.0, 9.5, 10.5, 12.0).

To measure the TG activity, the above-mentioned method of using ¹⁴C-labeled putrescine and dimethylcasein as the substrates was employed.Each buffer was added to the reaction system at a concentration of 50mM. As the enzyme source, used was the previously-prepared pure TGfraction at a concentration of 2 μg/ml. The reaction was conducted at37° C. for 30 minutes.

The relative values of the enzymatic activity were measured, inaccordance with the varying pH values of the individual reactionsystems. At pH of 8.2 (when Tris-HCl with pH of 8.5 was used as thebuffer), the reaction system showed the highest TG activity, which wasreferred to as a standard of 100. The results are shown in FIG. 2.

It has been found that the pH range suitable for the TG of the presentinvention is from about 7 to about 9, strictly from about 7.7 to about8.8 (see FIG. 2.). On the other hand, the pH range suitable for theBacillus subtilis-derived TG as reported by the group of the New MexicoState University is 9.5 or higher. Therefore, the TG as reported by themis obviously different from the Bacillus-derived TG of the presentinvention.

The temperature-dependent variation in the enzymatic activity of the TGwas measured according to the method mentioned below, from which thetemperature range suitable for the TG was determined.

To measure the TG activity, employed was the above-mentioned method ofusing ¹ 4C-labeled putrescine and dimethylcasein as the substrates.Concretely, the pH of the reaction system was adjusted to 7.5, using 0.1M Tris-HCl. As the enzyme source, the previously-prepared pure TGfraction was added to the reaction system at a concentration of 2 μg/ml.The reaction system was reacted for 30 minutes in a bath at temperaturesvarying from 25° C. to 80 C.

The relative values of the enzymatic activity were measured inaccordance with the varying reaction temperatures. At 60° C., thereaction system showed the highest TG activity, which was referred to asa standard of 100. The results are shown in FIG. 3.

It has been found that the temperature range suitable for the TG of thepresent invention is from about 40° C. to about 65° C., more strictlyfrom about 50° C. to about 62° C. (see FIG. 3).

Example 3 Determination of temperature stability of TG

The temperature-dependent stability of the TG of the invention wasmeasured according to the method mentioned below.

The previously-prepared pure TG fraction was reacted for 10 minutes in abath at temperatures varying from 10° C. to 80° C. After this, the TGactivity in each system was measured in the same manner as in Example 2.The relative values of the enzymatic activity were measured inaccordance with the varying reaction temperatures. The highest TGactivity as shown by the reaction system was referred to as a standardof 100. The results are shown in FIG. 4.

It has been found that the TG of the present invention is stable attemperatures not higher than about 60° C. (see FIG. 4).

Example 4 Crosslinking of protein with TG

The protein-crosslinking activity of the TG of the invention wasmeasured according to the method mentioned below.

A reaction system comprising α-casein at a final concentration of 1mg/ml, 0.1 M Tris-HCl (pH 7.5), 5 mM dithiothreitol and 5 mM sodiumazide was prepared.

The α-casein used herein was a commercial product produced by Sigma Co.To this system, added was the previously-prepared pure TG fraction at afinal concentration of 440 μg/ml.

The reaction system was reacted for 18 hours in a bath at 37° C. Thethus-reacted system was subjected to SDS-PAGE, which gave an additionalband at a higher polymer side in addition to the band for the substrate,α-casein. This indicates the cross-linking polymerization of α-casein.The results are shown in FIG. 5.

These results have verified the protein-crosslinking activity of the TGof the present invention (see FIG. 5).

The same results as above were also obtained when the TG of the presentinvention was reacted on bovine serum albumin (BSA). That is, thesubstrate protein BSA was also crosslinked by the TG of the invention.

Example 5 Effects of various reagents on TG activity

Effects, if any, of various reagents on the TG activity were checked.The reagents tested herein were N-ethylmaleimide (NEM), cystamine,phenylmethanesulfonyl fluoride (PMSF), ammonium sulfate, sodium sulfate,EDTA, EGTA, calcium chloride, dithiothreitol (DTT) and 2-mercaptoethanol(2-ME). All were bought from Nacalai Tesque, Inc.

As the enzyme source, the previously-prepared pure TG fraction was usedat a concentration of 2 μg/ml. The protein concentration was measured,using a protein assay kit (produced by Bio Rad Co.).

To measure the TG activity, the above-mentioned method of using ¹4C-labeled putrescine and dimethylcasein as the substrates (at pH of 7.5and at 37° C.) was employed. As the enzyme source, thepreviously-prepared pure TG fraction was used at a concentration of 2μg/ml. The reaction was conducted at 37° C. for 30 minutes.

The TG activity was measured in the following manner. Theabove-mentioned pure TG fraction was mixed with each one of the reagentsthat had been adjusted to have a suitable concentration and then left onice for 30 minutes. The solution of the enzyme source thus reacted witheach reagent was adjusted to have a TG concentration of 2 μg/ml and thenreacted with the substrates, whereupon the TG activity still remained inthe resulting reaction system was measured (at pH of 7.5 and at 37° C.).

The TG activity of the TG fraction that had not been reacted with anyone of the reagents was referred to as 100 (as control), and the TGactivity as remained in the reaction system that had been reacted witheach reagent was obtained as a relative value to the control. Theresults are shown in Table 1 below.

    ______________________________________                                                               Remaining Activity                                     Agent       Concentration                                                                            (relative value)                                       ______________________________________                                        --          --         100                                                    NEM         1           17                                                    cystamine   2           0                                                     PMSF        5           89                                                    (NH.sub.4).sub.2 SO.sub.4                                                                 10          1                                                     Na.sub.2 SO.sub.4                                                                         10          87                                                    EDTA        10         107                                                    EGTA        10          98                                                    CaCl.sub.2  5           65                                                    DTT         10         119                                                    2-ME        10         111                                                    ______________________________________                                    

The TG activity of the TG of the present invention was inhibited by NEM,while it was not inhibited by the reducing agents of DTT and 2-ME butwas rather enhanced by these to some degree. These results suggest theprobability that the cysteine residue will participate in the expressionof the TG activity.

The TG of the present invention was not inhibited by DTT, while theBacillus subtilis-derived TG as reported by the group of the New MexicoState University is known to have been inhibited by DTT. That is, theboth TGs have obviously different properties.

In addition, the TG of the present invention was not inhibited by sodiumsulfate but was inhibited by ammonium sulfate and cystamine.Accordingly, it has been clarified that the TG of the present inventionis characterized in that its activity is inhibited in reaction systemscontaining some amines.

Moreover, the TG of the present invention was not inhibited by thechelating agents of EGTA and EDTA. In view of this property of it, theTG of the present invention is different from the TG as reported by thegroup of the New Mexico State University. In other words, it can be saidthat the TG of the present invention does not have the property ofrequiring metal ions such as Ca²⁺, etc. The reaction systems containingthe TG of the present invention, which were used herein for measuringthe TG activity, did not contain Ca²⁺, and the TG of the presentinvention exhibited the TG activity in these Ca²⁺ -free reactionsystems. From the results, it can be concluded that the TG of thepresent invention is independent of Ca²⁺.

Furthermore, the TG of the present invention maintained the TG activityof not less than 50% in the presence of Ca2+ of not less than 5 mM.Thus, the TG of the present invention is different from the TG asreported by the group of the New Mexico State University in that thelatter is known to have been greatly inhibited by Ca²⁺ of 5 mM or more(see Table 1).

Example 6

TG derived from Bacillus subtilis AJ12866 strain was purified, and itsproperties were determined.

Cells of Bacillus subtilis AJ12866 were incubated in a Schaeffer'smedium at 37° C. for 16 hours by shaking cultivation. 3 ml of theculture was added to 30 ml of another Schaeffer's medium and furtherincubated therein at 37° C. for 12 hours. The Schaeffer's media used hada composition comprising 8 g/liter of Bacto-nutrient broth, 1 g/liter ofKCl, 0.12 g/liter of MgSO₄.7H₂ O, 1 mM of CaCl₂, 10 μM of MnCl₂ and 1 μMof FeSO₄ and had pH of 7.0.

The culture was centrifuged at 10,000×g for 20 minutes to separate theprecipitate from the supernatant.

The precipitate was ground, using glass beads. The TG activity of eachof the culture supernatant, the culture precipitate and the groundprecipitate liquid (cell debris liquid) was measured. The method formeasuring the enzymatic activity as employed herein was as follows. 50μl of a reaction system (100 mM Tris-HCl, pH 7.5, 6.3 mg/mldimethylcasein, 10 nM ¹⁴ C-putrescine, 1.2 μCi) containing 10 μl of theenzyme sample to be measured was kept at 37° C. for 30 minutes therebyto make the substrates reacted. After the reaction, 40 μl of thereaction mixture was adsorbed onto filter paper. In the reactionmixture, the putrescine was reacted with the dimethylcasein due to thecatalysis of the TG therein. A bonded reaction product of the putrescineand the dimethylcasein was adsorbed on the filter paper. By adding 10%TCA thereto, the bonded reaction product was fixed on the filter paper.Then, the filter paper was washed three times with a 5% TCA solution,and the ¹ 4C radioactivity as fixed to the filter paper was counted witha liquid scintillation counter. The value thus measured corresponds tothe relative TG activity of the enzyme sample. The results obtained areshown in Table 2 below.

                  TABLE 2                                                         ______________________________________                                                       Relative Activity                                              ______________________________________                                        culture supernatant                                                                            280                                                          culture precipitate                                                                            510                                                          ground precipitate liquid                                                                      10200                                                        ______________________________________                                    

These results show that the ground precipitate liquid sample which is afraction containing the bacterial spores has a much higher TG activitythan the other samples.

Example 7 Determination of the stage at which the Bacillus-derived TG isinduced

Cells of Bacillus subtilis AJ12866 strain were incubated in the samemanner as in Example 6, while the TG activity of the cell debrissuspension as sampled was measured at given intervals. For thepreparation of the cell debris suspension and the measurement of the TGactivity, the processes of Example 1 and Example 6 were referred to. Thedegree of growth of the cells of Bacillus subtilis AJ12866 beingincubated was represented by the degree of turbidity of the culture. Todetermine the degree of turbidity of the culture, a ray having awavelength of 610 nm was applied to the culture and the absorbance ofthe ray was measured. The results obtained are shown in FIG. 6. As isknown from these results, the TG activity of the culture began toincrease after the growth of the cells reached the stage of stationarygrowth and the cells began to form their spores (about 4 hours after thestart of the incubation).

Example 8 Purification of Bacillus-derived TG

Using the cells as incubated in the same manner as in Example 7, thefollowing experiments were carried out.

The cells of Bacillus subtilis AJ12866 strain were suspended in areaction system (0.5 mg/ml lysozyme, 20 μg/ml DNase I, 0.1 M Tris-HCl(pH 7.5), 2 mM DTT, 1 mM EDTA, 2 mM PMSF) and reacted on ice for 2 hourswhereby the cells were lysed. The resulting reaction mixture wascentrifuged at 20,000×g for 20 minutes, and the precipitate fractionthus obtained was suspended in a washing system (0.1 M Tris-HCl (pH7.5), 1 mM EDTA, 2 mM PMSF). The resulting suspension was centrifuged,and the precipitate was collected. This operation was repeated for atotal of two times.

The thus-obtained precipitate fraction was suspended in a buffer (0.1 Msodium carbonate (pH 10), 1 mM EDTA, 2 mM PMSF) and left at 37° C. for30 minutes. Meanwhile, a part of the substances that had existed in theprecipitate fraction were dissolved in the buffer and the TG activitywas transferred to the soluble fraction. This was then centrifuged andthe resulting supernatant exhibited TG activity. The pH of thesupernatant was adjusted to 6.0 by adding acetic acid thereto. This isreferred to as a crude enzyme liquid. The crude enzyme liquid wasconcentrated by ultrafiltration and then dialyzed against a buffer (50mM Tris-HCl (pH 7.5), 0.1 M NaCl), whereby the buffer was exchanged. Theresulting crude enzyme liquid was subjected to gel permeation, and theTG active fraction thus eluted was collected. The enzymatic propertiesof the fraction were measured.

The results revealed the following facts. For the measurement of theproperties, the processes of Examples 1 through 5 were referred to.

(1) The pH range suitable for the TG was from about 7 to about 9 (seeFIG. 7). (2) The temperature range suitable for the TG was from about40° C. to about 65° C. (see FIG. 8). (3) Regarding its temperaturestability, the TG was stable at about 60° C. or lower (see FIG. 9). (4)The TG was greatly inhibited by cystamine, NEM and (NH₄)₂ SO₄ (see FIG.10 and Table 3).

                  TABLE 3                                                         ______________________________________                                                    Concentration                                                                            Remaining Activity                                     Agent       (mM)       (Relative value)                                       ______________________________________                                        --          --         100                                                    PMSF         5          95                                                    (NH.sub.4).sub.2 SO.sub.4                                                                 10          3                                                     Na.sub.2 SO.sub.4                                                                         10          85                                                    EGTA        10          94                                                    2-ME        10         122                                                    ______________________________________                                    

(5) The TG activity was not inhibited by DTT but rather increased byabout 1.5 times or more in the presence of 1 mM of DTT (see FIG. 11).(6) EDTA had almost no influence on the TG activity (see FIG. 12). (7)The TG activity was not inhibited by Ca²⁺ of 5 mM or more. Theexpression of the TG activity does not require the presence of Ca²⁺ions. In other words, the TG is independent of Ca²⁺ (see FIG. 13). (8)The TG had a molecular weight of (a) from about 18,000 to about 22,000(as measured by gel permeation) and (b) from about 28,000 to about30,000 (as measured by SDS-PAGE).

In addition, the amino acid sequence of the TG near its N-terminal or,namely, the amino acid sequence thereof of 35 residues from itsN-terminal was determined. The result revealed the high homology of theTG obtained herein to the Bacillus subtilis AJ1307-derived TG obtainedpreviously. Precisely, the amino acid sequences of the two TGs aredifferent from each other only in the 22nd amino acid residue. TheAJ1307-derived TG has asparagine residue at the 22nd amino acid residue,while the AJ12866-derived TG has aspartic acid residue at the 22nd aminoacid residue.

The results of the above-mentioned experiments show that the TG derivedfrom Bacillus subtilis AJ12866 has the same properties as those of theabove-mentioned, Bacillus subtilis AJ1307-derived TG.

Example 9 Gellation of Proteins with TG of the invention

The TG active fraction as obtained after the gel permeation in Example 8was mixed with a 10% casein solution (25 mM Tris-HCl (pH 7.5), 5 mM DTT)at a ratio of 1:9 and reacted at 37° C. for 24 hours. After thereaction, the solution was gelled.

The pure TG as obtained in Example 1 was added to a 7% gelatin solutionat a concentration of 2 units/g-protein and reacted at 35° C. for 2hours. After the reaction, the gelatin protein solution was gelled.

Example 10 Isolation of Bacillus-derived TG gene

(1) Purification of TG and Determination of its N-terminal amino acidsequence:

The partial amino acid sequence of the Bacillus-derived TG as determinedin Example 1, which corresponds to SEQ ID NO:1 in the Sequence List, waschecked as to whether or not it is homologous to any amino acidsequences of known peptides. However, there was found no homology of theformer to any amino acid sequences as registered in GenBank (LASL-GDB),SWISS-PROT and NBRF(PIR).

The amino acid sequence of the TG was back-translated on the basis ofthe universal codon, from which the base sequence that codes for theamino acid sequence was deduced. Then, the base sequence was checked asto whether or not it is homologous to any base sequences of knownnucleic acids. The results revealed the presence of high homology of theformer to one base sequence as registered in GenBank (LASL-GDB). Thebase sequence that was found herein to be homologous to the basesequence of the TG of the invention has an accession number of L29189,and its source is D. W. Hanlon & G. W. Ordal, J. Biol. Chem., Vol. 269,pp. 14038-14046 (1994). This reference naturally discloses the basesequences of genes that code for the transmembrane receptor of Bacillussubtilis, and the base sequence which is disclosed therein and which wasnow found herein to be homologous to the base sequence of the TG of theinvention, is positioned in the upstream flanking region. Concretely,the base sequence of the TG of the invention was found to be homologousto the disclosed base sequence in question of from 1st to 68th baseresidues. The latter base sequence composed of 68 base pairs ispositioned in the 5'-upstream site of the mcpB gene of Bacillussubtilis, and its transcribing direction is opposite to that of the mcpBgene. The functions of the peptide as coded for by the sequence composedof 68 base pairs are not referred to by D. W. Hanlon & G. W. Ordal in J.Biol. Chem. Vol. 269, pp. 14038-14046 (1994).

(2) Collection of cells:

Cells of Bacillus subtilis AJ1307 strain were incubated under theconditions mentioned below. Using Schaeffer's medium, the incubation wasconducted always at 37° C. by liquid shaking cultivation. First, cellsof AJ1307 strain were incubated in 20 ml of a Schaeffer's mediumovernight for seed cultivation. 5 ml of the culture comprising the seedcells was finally incubated in 100 ml of another Schaeffer's medium.

(3) Isolation of chromosome DNA from cells:

After the cells reached the latter stage of logarithmic growth phaseunder the conditions mentioned above, 100 ml of the culture wascentrifuged (at 12,000×g, at 4° C. and for 15 minutes) and the cellswere collected. The cells were suspended in 10 ml of 50:20 TE (50 mMTris-HCl, pH 8.0, 20 mM EDTA), washed and centrifuged to again collectthe cells. And, again, the cells were suspended in 10 ml of 50:20 TE.0.5 ml of a 20 mg/ml lysozyme solution and 1 ml of a 10% SDS solutionwere added to the resulting suspension, in which the cells wereincubated at 55° C. for 20 minutes. After the incubation, a 10:1TE-saturated phenol of the same volume as that of the suspension wasadded to the suspension, whereby the suspension was de-proteined. To thethus-separated aqueous layer, 2-propanol of the same volume as that ofthe layer was added, by which the DNA was precipitated and collected.The thus-obtained DNA precipitate was dissolved in 0.5 ml of 50:20 TE,and then 5 μl of 10 mg/ml RNase and 5 μl of 10 mg/ml Proteinase K wereadded thereto and reacted at 55° C. for 2 hours. After the reaction, a10:1 TE-saturated phenol of the same volume as that of the solution wasadded to the solution, by which the solution was de-proteined. To thethus-separated aqueous layer, added was 24:1 chloroform/isoamyl alcoholof the same volume as that of the layer and stirred, and then theaqueous layer was collected. This operation was repeated for a total oftwo times. To the final aqueous layer thus obtained, were added a 3 Msodium acetate solution (pH 5.2) at a final concentration of 0.4 M andethanol of twice the volume of the layer. The DNA as precipitated wascollected, washed with 70% ethanol, dried and then dissolved in 1 ml of10:1 TE.

(4) Preparation of DNA fragment by PCR:

To isolate and amplify the DNA molecule containing the gene that codesfor the Bacillus-derived TG, employed was TAKARA LA PCR IN VITRO CLONINGKIT (produced by Takara Shuzo Co.). Unless otherwise specificallyindicated below, the following experiments were carried out according tothe method as instructed in the specification attached to the kit.

5 μg of the chromosome DNA as prepared in the previous process (3) wasdigested with a restriction enzyme Hind III. Next, the DNA fragment wascollected by ethanol precipitation, to which was linked a Hind IIIcassette. This was again subjected to ethanol precipitation, and thecollected DNA was subjected to the first PCR using Primer C1 and PrimerS1. The base sequence of Primer C1 used corresponds to SEQ ID NO:4 inSequence List, and that of Primer S1 to SEQ ID NO:5 in the same. PrimerC1 is contained in the TaKaRa LA PCR in vitro Cloning Kit used, of whichthe base sequence is within the base sequence of the Hind III cassette.The base sequence of Primer S1 is complementary to the region of fromthe 566th guanosine residue to the 600th adenosine residue in the basesequences of the above-mentioned genes that code for the transmembranereceptor of Bacillus subtilis.

The PCR reaction was conducted for a total of 30 cycles under theconditions mentioned below, using GENEAMP PCR SYSTEM 9600 (produced byPerkin Elmer Co.).

One PCR cycle:

98° C., 20 seconds

68° C., 3 minutes

Next, the reaction mixture was diluted to 1/100, to which were addedPrimer C2 and Primer S2 for the 2nd PCR. The conditions for the 2nd PCRwere the same as those for the 1st PCR. The base sequence of Primer C2and that of Primer S2 correspond to SEQ ID NO:6 and SEQ ID NO:7,respectively, in the Sequence List. Primer C2 is contained in the TAKARALA PCR IN VITRO CLONING KIT used, of which the base sequence is withinthe base sequence of the Hind III cassette. The base sequence of PrimerS2 is complementary to the region of from the 34th thymidine residue tothe 68th thymidine residue in the base sequences of the above-mentionedgenes that code for the transmembrane receptor of Bacillus subtilis.

After the reaction, 3 μl of the reaction mixture was subjected to 0.8%agarose gel electrophoresis, which verified that a DNA fragment of about2 kb had been amplified.

(5) Cloning of the PCR-amplified DNA fragment with pUC18:

The PCR-amplified DNA fragment of about 2 kb was cloned by ligating itwith pUC18. The cloning was conducted using SURE CLONE LIGATION KIT(produced by Pharmacia Co.). Unless otherwise specifically indicatedbelow, the following experiments were carried out according to themethod as instructed in the specification attached to the kit. 400 ng ofthe thus-amplified DNA fragment of about 2 kb were processed to make theboth ends thereof blunt, and then phosphorylated. After thephosphorylation, the DNA fragment was purified and then ligated withPUC18 that had been digested with SmaI. Using the reaction liquid thuscontaining the ligated DNA fragment, cells of Escherichia coli weretransformed with the DNA.

From the cells thus transformed, some JMlO9 transformants asintentionally transformed with the pUC18 containing the DNA fragment ofabout 2 kg were selected by screening. For the screening, referred towas the screening method described in Molecular Cloning, 2nd Edition,Cold Spring Harbor Press (1989).

(6) DNA sequencing of TG gene:

The plasmid which the screened transformants had was prepared inaccordance with the method described in Molecular Cloning, 2nd Edition,Cold Spring Harbor Press (1989), from which the base sequence of theamplified DNA fragment of about 2 kb was determined. The sequencing wasconducted, using DYE TERMINATOR CYCLE SEQUENCING KIT (produced by ABICo.) and according to the method as instructed in the specificationattached to the kit. The electrophoresis was conducted, using DNASEQUENCER 373 (produced by ABI Co.).

The sequenced result revealed that the PCR-amplified DNA fragment has aregion of from 118th adenosine residue to the 1042nd thymidine residueof the base sequence of SEQ ID NO:2 in the Sequence List. Of the basesequence of SEQ ID NO:2, the region of from 118th adenosine residue tothe 852th cytosine residue is the open reading frame. The amino acidsequence of the polypeptide which the ORF codes for can be presumed onthe basis of the universal codon. The amino acid sequence in question isshown below along with the base sequence of SEQ ID NO:2.

Of the amino acid sequence, the region of from its N-terminal to the35th amino acid residue is entirely the same as the amino acid sequencecomposed of 35 amino acid residues as referred to in the previousprocess (1). This demonstrates that the PCR-amplified DNA fragment isthe intended Bacillus-derived TG gene.

It could be concluded that the difference between the base sequence ofSEQ ID NO:2 and that as disclosed by D. W. Hanlon & G. W. Ordal in J.Biol. Chem., Vol. 269, pp. 14038-14046 (1994) would be due to thedifference in the strains used between them.

The plasmid having the Bacillus-derived TC gene as inserted thereunto inthe direction in which the pUC18-derived lac promoter acts on the genefor transcription is referred to as pBSTG75-11.

Example 11 Cloning of Bacillus-derived TG gene from chromosome DNAlibrary.

(1) Formation of chromosome DNA library:

1 μg of the chromosome DNA as prepared in Example 10 was completelydigested with Hind III. The DNA was recovered by ethanol precipitationand then dissolved in 10 μl of 10:1 TE. 5 μl of the resulting solutionwas mixed with 1 ng of pUC118 (produced by Takara Shuzo Co.) that hadbeen digested with Hind III and then dephosphorylated with BAP, and theDNA was ligated with the pUC118, using DNA LIGATION KIT VER. 2 (producedby Takara Shuzo Co.). 100 μl of competent cells of Escherichia coliJM109 strain (produced by Takara Shuzo Co.) were mixed with 3 μl of thethus-ligated reaction mixture, and the cells of Escherichia coli JM109strain were transformed with the DNA. The transformant cells were coatedover a suitable solid medium to prepare a chromosome DNA library.

(2) Formation of probe:

As the probe, used was the whole length of the TG gene as obtained inExample 1. Using pBSTG75-11 as the template, this was subjected to PCRwith Primer S2 and Primer S3. The PCR was conducted, using TAKARA LA PCRKIT Ver. 2.

100 μl of a reaction system comprising 10 ng of the template,pBSTG75-11, 20 pmol of Primer S2 and 20 pmol of Primer S3 was preparedand reacted. Primer S3 is a 35-mer that is complementary to the regionof 35 bases of from 818th base to the 852nd base of the base sequence(SEQ ID NO:2) of the TG gene, and its base sequence corresponds to SEQID NO:8 in the Sequence List. The PCR was conducted for a total of 30cycles under the following conditions.

One PCR cycle:

94° C., 30 seconds

55° C., 30 seconds

72° C., one minute

The DNA fragment thus amplified by the above-mentioned reaction wasseparated by electrophoresis with 1% agarose gel (SEAPLAQUE GTG,produced by FMC Co.). The intended band was cut out, and the DNA waspurified therefrom, using EASY PREP SYSTEM (produced by Pharmacia Co.)and PCR PRODUCTS PREP KIT (produced by Pharmacia Co.). Thus finally, 200μl of a DNA (4 ng/μl) solution was obtained.

The DNA fragment was labeled with ³² p and used as the probe. UsingRANDOM PRIMER DNA LABELING KIT Ver. 2 (produced by Takara Shuzo co.),the probe was labeled with α-³² P!dCTP (3000 μCi/mmol) (produced byAmersham Co.), in accordance with the method as instructed in thespecification attached to the kit.

(3) Colony hybridization:

One example of colony hybridization is described in Molecular Cloning,2nd Edition, Cold Spring Harbor Press (1989), which was referred tobelow.

Colonies of the chromosome DNA library were transferred onto a nylonmembrane filter (HYBOND-N, produced by Amersham Co.), which was thenalkali-denatured, neutralized and fixed.

The hybridization was conducted, using RAPID-HYB BUFFER (produced byAmersham Co.). Concretely, the filter was dipped in the buffer andsubjected to prehybridization at 65° C. for 4 hours. After this, thelabeled probe as prepared in the previous process (2) was added to thebuffer, in which the hybridization was conducted at 65° C. for 2 hours.Next, the filter was washed with 2×SSC containing 0.1% SDS at roomtemperature for 20 minutes. Further, this was washed twice with 0.1×SSCcontaining 0.1% SDS at 65° C. for 15 minutes.

The results of this process verified the production of five coloniesthat hybridized with the probe.

(4) DNA sequencing of TG gene:

In the same manner as in Example 5, the base sequence of the DNAfragment that had been inserted into pUC118 was determined. The resultof this sequencing verified that the DNA has the base sequence of SEQ IDNO:2 in the Sequence List.

Example 12 Expression of Bacillus-derived TG gene in Escherichia coli:

(1) Incubation of cells of Escherichia coli having recombinant TG geneand expression and induction of the recombinant TG gene in the cells:

The plasmid pBSTG75-11 as obtained in Example 10 has a DNA coding forthe Bacillus-derived TG along with a DNA coding for a lacZ protein insuch a way that the former DNA has been linked to the downstream site inthe latter DNA therein. From its base sequence, it is presumed that theplasmid would be designed to be able to express a fused protein with apeptide having an amino acid sequence of SEQ ID NO:9 in the SequenceList, which is composed of 11 amino acid residues and which has beenadded to the Bacillus-derived TG before the first methionine residue ofthe sequence of the TG.

In this experiment, test cells of Escherichia coli JM109 transformedwith pBSTG75-11 and control cells of Escherichia coli JM109 transformedwith pUC18 were used. The test cells and the control cells wereseparately incubated in an LB medium containing 100 mg/ml of ampicillin,at 37° C. by liquid shaking cultivation. Concretely, the cells wereimplanted in 30 ml of the medium and incubated overnight by shakingcultivation to prepare seed cultures. On the other hand, four flaskseach containing 30 ml of a fresh medium were prepared. The seed cultureof the test cells of Escherichia coli JM109 transformed with pBSTG75-11was implanted in two flasks at a cell concentration of 5%. The twoflasks were referred to as Test Group 1 and Test Group 2. On the otherhand, the seed culture of the control cells of Escherichia coli JM109transformed with pUC18 was implanted in the other two flasks at a cellconcentration of 5%. The two flasks were referred to as Test Group 3 andTest Group 4. The cells in each group were incubated. After theabsorbance at 610 nm became about 0.7, IPTG was added to only Test Group1 and Test Group 3 at a final concentration of 1 mM. Four hours afterthis, the incubation of the cells in every group was terminated.

(2) Confirmation of protein induced and expressed:

After the termination of the incubation, the cells in each culture wereobserved with a microscope. The results showed that only the cells JM109transformed with pBSTG75-11, to which IPTG had been added, contained aprotein inclusion body in themselves.

After the termination of the incubation, 10 ml of each culture wascentrifuged (at 12,000×g for 15 minutes) to collect the cells. The cellswere suspended in 2 ml of 10 mM Tris-HCl (pH 7.5), washed and againcentrifuged to collect the cells. The cells were suspended in 1 ml ofthe same buffer and disrupted by shaking the resulting suspension alongwith 0.1 mm zirconia beads for 3 minutes, using MINI-BEAD BEATER(produced by Wakenyaku Co.). The thus-disrupted suspension was subjectedto SDS-PAGE and stained with CBB. The results showed that the suspensionof Test Group 1 (cells of JM109 transformed with pBSTG75-11 and inducedby IPTG) gave a band of from about 29,000 to about 30,000. From themolecular weight thus identified, it was presumed that the cells of TestGroup 1 would express the expected fused protein.

(3) Confirmation of TG activity:

The TG activity of the expressed protein was measured. Concretely, 10 μlof the previously-prepared suspension that contained disrupted cells wasadded to a reaction system containing dimethylcasein and ¹⁴ C-labeledputrescine and reacted. After the reaction, the reaction mixture wasadsorbed onto filter paper, and the amount of the putrescine as caughtby dimethylcasein on the filter paper was measured, using a liquidscintillation counter. The results are shown in Table 4 below.

                  TABLE 4                                                         ______________________________________                                        Harboured Plasmid                                                                          Induction w/ IPTG                                                                          TG activity (dpm)                                   ______________________________________                                        pBSTG75-11   +            496                                                 Test Group 1                                                                  pBSTG75-11   -            0                                                   Test Group 2                                                                  pUC18        +            0                                                   Test Group 3                                                                  pUC18        -            0                                                   Test Group 3                                                                  ______________________________________                                    

These results verified that the cells JM109 transformed with pBSTG75-11and induced by IPTG (in Test Group 1) exhibited TG activity.

The Escherichia coli KM109 strain transformed with pBSTG75-11 wasreferred to as Escherichia coli AJ13172, which was deposited with theinternational depository in the Bioengineering Laboratory on Dec. 20,1995 under the provisions of the Budapest Treaty, under internationaldeposit number FERM BP-5446.

It has now been confirmed that the DNA having the base sequence of SEQID NO:2 in the Sequence List codes for an enzyme having TG activity.Namely, it has now been clarified that this DNA has a Bacillus-derivedTG gene. In addition, it has also been confirmed that, even whenadditional DNA that codes for a different peptide is added to this DNA,the resulting combination DNA can express the Bacillus-derived TG gene,and that the fused protein to be expressed by the combination DNA has TGactivity. Moreover, it has been clarified that the fused protein can beintracellularly expressed by the intentionally-transformed cells ofEscherichia coli as a protein inclusion body and that the expression ofthe protein can be controlled by suitable promoters.

Having now fully described the invention, it will be apparent to one ofordinary skill in the art that many changes and modifications can bemade thereto without departing from the spirit or scope of the inventionas set forth herein.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 9                                             - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 35 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 - Met Ile Ile Val Ser Gly Gln Leu Leu Arg Pr - #o Gln Asp Ile Glu Asn         #                15                                                           - Trp Gln Ile Asp Gln Asn Leu Asn Pro Leu Le - #u Lys Glu Met Ile Glu         #            30                                                               - Thr Pro Val                                                                         35                                                                    - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 1042 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 118..843                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 - CTGCTTAAAA AGTTTTAAAA TAAAAAATGG AAGAAGTTCT TTTTGGCAGT CT - #TCTGTCTT         60                                                                          - TTTAGCTTTC ATTGCCCAAG CTCTTTGCAT ATCTTATATA AACAAGGGGG GC - #TAAAC           117                                                                          - ATG ATT ATT GTA TCA GGA CAA TTG CTC CGT CC - #C CAG GAT ATT GAA AAT          165                                                                          Met Ile Ile Val Ser Gly Gln Leu Leu Arg Pr - #o Gln Asp Ile Glu Asn           #                 15                                                          - TGG CAG ATT GAT CAA AAT CTG AAT CCG CTG TT - #A AAA GAG ATG ATT GAG          213                                                                          Trp Gln Ile Asp Gln Asn Leu Asn Pro Leu Le - #u Lys Glu Met Ile Glu           #             30                                                              - ACG CCT GTT CAG TTT GAT TAT CAT TCA ATT GC - #T GAA CTG ATG TTT GAG          261                                                                          Thr Pro Val Gln Phe Asp Tyr His Ser Ile Al - #a Glu Leu Met Phe Glu           #         45                                                                  - CTT AAA CTG CGG ATG AAT ATT GTA GCA GCG GC - #A AAG ACG CTG CAC AAA          309                                                                          Leu Lys Leu Arg Met Asn Ile Val Ala Ala Al - #a Lys Thr Leu His Lys           #     60                                                                      - AGC GGG GCG AAG TTT GCC ACT TTT TTA AAA AC - #A TAC GGG AAT ACA ACG          357                                                                          Ser Gly Ala Lys Phe Ala Thr Phe Leu Lys Th - #r Tyr Gly Asn Thr Thr           # 80                                                                          - TAT TGG AGG GTT TCA CCG GAG GGC GCC TTG GA - #G CTG AAA TAC AGA ATG          405                                                                          Tyr Trp Arg Val Ser Pro Glu Gly Ala Leu Gl - #u Leu Lys Tyr Arg Met           #                 95                                                          - CCG CCT TCA AAA GCG ATT CGG GAC ATT GCA GA - #G AAC GGC CCG TTT TAT          453                                                                          Pro Pro Ser Lys Ala Ile Arg Asp Ile Ala Gl - #u Asn Gly Pro Phe Tyr           #           110                                                               - GCG TTT GAA TGC GCA ACC GCA ATC GTT ATC AT - #T TAT TAC TTG GCC TTA          501                                                                          Ala Phe Glu Cys Ala Thr Ala Ile Val Ile Il - #e Tyr Tyr Leu Ala Leu           #       125                                                                   - ATC GAT ACA ATC GGT GAA GAT AAA TTC AAT GC - #C AGC TTT GAC AGA ATT          549                                                                          Ile Asp Thr Ile Gly Glu Asp Lys Phe Asn Al - #a Ser Phe Asp Arg Ile           #   140                                                                       - ATT TTA TAT GAC TGG CAT TAT GAG AAA TTG CC - #G ATC TAT ACG GAA ACA          597                                                                          Ile Leu Tyr Asp Trp His Tyr Glu Lys Leu Pr - #o Ile Tyr Thr Glu Thr           145                 1 - #50                 1 - #55                 1 -       #60                                                                           - GGA CAC CAC TTT TTC CTT GGA GAT TGT TTG TA - #T TTT AAG AAT CCT GAA          645                                                                          Gly His His Phe Phe Leu Gly Asp Cys Leu Ty - #r Phe Lys Asn Pro Glu           #               175                                                           - TTT GAT CCG CAA AAG GCG CAA TGG AGA GGC GA - #A AAT GTG ATT TTA CTG          693                                                                          Phe Asp Pro Gln Lys Ala Gln Trp Arg Gly Gl - #u Asn Val Ile Leu Leu           #           190                                                               - GGG GAA GAT AAA TAT TTT GCC CAT GGT CTT GG - #A ATC TTA AAC GGA AAG          741                                                                          Gly Glu Asp Lys Tyr Phe Ala His Gly Leu Gl - #y Ile Leu Asn Gly Lys           #       205                                                                   - CAA ATT ATA GAT AAG CTG AAT TCT TTT AGG AA - #A AAA GGA GCC TTA CAG          789                                                                          Gln Ile Ile Asp Lys Leu Asn Ser Phe Arg Ly - #s Lys Gly Ala Leu Gln           #   220                                                                       - TCA GCC TAC CTT CTG TCT CAG GCG ACC AGA CT - #G GAT GTT CCG TCT CTT          837                                                                          Ser Ala Tyr Leu Leu Ser Gln Ala Thr Arg Le - #u Asp Val Pro Ser Leu           225                 2 - #30                 2 - #35                 2 -       #40                                                                           - TTC CGC ATCGTCCGCT AAAAAGCCCC ATCGCCTATT TTCGGGACGA TG - #GGGTTTCA           893                                                                          Phe Arg                                                                       - AATGCCTTTC GTTTTCGATA GAAGGGGGCT GTGCCGAAAT ATTGGTTCGC AG - #CCCACTCC        953                                                                          - ATTTTTTCAA GGTCATTTCT TGTCACGATT GGATCCTGGC TGCTCCATTT GA - #TAAAGCGG       1013                                                                          #          1042    TGAT AGGAACCAT                                             - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 242 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 - Met Ile Ile Val Ser Gly Gln Leu Leu Arg Pr - #o Gln Asp Ile Glu Asn         #                 15                                                          - Trp Gln Ile Asp Gln Asn Leu Asn Pro Leu Le - #u Lys Glu Met Ile Glu         #             30                                                              - Thr Pro Val Gln Phe Asp Tyr His Ser Ile Al - #a Glu Leu Met Phe Glu         #         45                                                                  - Leu Lys Leu Arg Met Asn Ile Val Ala Ala Al - #a Lys Thr Leu His Lys         #     60                                                                      - Ser Gly Ala Lys Phe Ala Thr Phe Leu Lys Th - #r Tyr Gly Asn Thr Thr         # 80                                                                          - Tyr Trp Arg Val Ser Pro Glu Gly Ala Leu Gl - #u Leu Lys Tyr Arg Met         #                 95                                                          - Pro Pro Ser Lys Ala Ile Arg Asp Ile Ala Gl - #u Asn Gly Pro Phe Tyr         #           110                                                               - Ala Phe Glu Cys Ala Thr Ala Ile Val Ile Il - #e Tyr Tyr Leu Ala Leu         #       125                                                                   - Ile Asp Thr Ile Gly Glu Asp Lys Phe Asn Al - #a Ser Phe Asp Arg Ile         #   140                                                                       - Ile Leu Tyr Asp Trp His Tyr Glu Lys Leu Pr - #o Ile Tyr Thr Glu Thr         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Gly His His Phe Phe Leu Gly Asp Cys Leu Ty - #r Phe Lys Asn Pro Glu         #               175                                                           - Phe Asp Pro Gln Lys Ala Gln Trp Arg Gly Gl - #u Asn Val Ile Leu Leu         #           190                                                               - Gly Glu Asp Lys Tyr Phe Ala His Gly Leu Gl - #y Ile Leu Asn Gly Lys         #       205                                                                   - Gln Ile Ile Asp Lys Leu Asn Ser Phe Arg Ly - #s Lys Gly Ala Leu Gln         #   220                                                                       - Ser Ala Tyr Leu Leu Ser Gln Ala Thr Arg Le - #u Asp Val Pro Ser Leu         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Phe Arg                                                                     - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 35 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "SYNTHETIC DNA, PRIMER C1desc                                                             FOR PCR"                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 #       35         GAAC GCGTAATACG ACTCA                                      - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 35 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "SYNTHETIC DNA, PRIMER S1desc                                                             FOR PCR"                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                 #       35         GTGC CGTTATCTGC GCCCC                                      - (2) INFORMATION FOR SEQ ID NO:6:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 35 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "SYNTHETIC DNA, PRIMER S2desc                                                             FOR PCR"                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                 #       35         ACGA CTCACTATAG GGAGA                                      - (2) INFORMATION FOR SEQ ID NO:7:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 35 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "SYNTHETIC DNA, PRIMER S2desc                                                             FOR PCR"                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                 #       35         GACA ATTGCTCCGT CCCCA                                      - (2) INFORMATION FOR SEQ ID NO:8:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 35 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "SYNTHETIC DNA, PRIMER S3desc                                                             FOR PCR"                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                 #       35         AGAG ACGGAACATC CAGTC                                      - (2) INFORMATION FOR SEQ ID NO:9:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 11 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                 - Met Thr Met Ile Thr Asn Ser Ser Ser Val Pr - #o                             #                10                                                           __________________________________________________________________________

What is claimed as new and is desired to be secured by Letters Patent ofthe United States is:
 1. A method for processing a protein, anon-proteinaceous amino acid polymer, or a peptide or derivativesthereof having a crosslinked structure, which comprises:contactingglutamine and lysine residues in a protein, a non-proteinaceous aminoacid polymer, a peptide or derivatives thereof with a transglutaminaseobtained from Bacillus subtilis to form intermolecular orintramolecular, crosslinked ε-(γ-Glu)-Lys bonds between or in themolecules of the protein, non-proteinaceous amino acid polymer, peptideor derivatives thereof, wherein said transglutaminase has the followingphysicochemical properties:a) it is active between about pH 7 and pH 9,b) it is active between about 40° C. and about 65° C., c) it is stableat about 60° C. or lower, d) the enzymatic activity of thetransglutaminase is not dependent on Ca⁺² and has an activity of 50% ormore in the presence of 5 mM of Ca⁺², e) it is inhibited byN-ethylmaleimide, cystamine or ammonium sulfate, f) it is not inhibitedby ethylenediamine-tetraacetic acid, dithiothreitol or2-mercaptoethanol, g) it has a molecular weight of (i) from about 18,000to about 22,000 as measured by gel permeation, and (ii) from about28,000 to about 30,000 as measured by SDS-PAGE, and h) it catalyzes thetransacylation of the γ-carboxyamide group in glutamine residue(s) in apeptide chain.
 2. A method for processing a protein, a non-proteinaceousamino acid polymer, a peptide or derivatives thereof having acrosslinked structure, which comprises:crosslinking glutamine and lysineresidues in a protein, a non-proteinaceous amino acid polymer, a peptideor derivatives thereof with a transglutaminase fraction isolated byfractionating spores obtained by disrupting or lysing sporangia ofBacillus subtilis, to thereby form intramolecular or intermolecular,crosslinked ε-(γ-Glu)-Lys bonds between or in the molecules of theprotein, non-proteinaceous amino acid polymer, peptide or derivativesthereof.
 3. A purified or isolated DNA coding for a transglutaminaseobtained from Bacillus subtilis having the following physicochemicalproperties:a) it is active between pH 7 and about pH 9, b) it is activebetween about 40° C. and about 65° C., c) it is stable at about 60° C.or lower, d) the enzymatic activity of the transglutaminase is notdependent on Ca⁺² and has an activity of 50% or more in the presence of5 mM of Ca⁺², e) it is inhibited by N-ethylmaleimide, cystamine orammonium sulfate, f) it is not inhibited by ethylenediamine-tetraaceticacid, dithiothreitol or 2-mercaptoethanol, g) it has a molecular weightof (i) from about 18,000 to about 22,000 as measured by gel permeation,and (ii) from about 28,000 to about 30,000 as measured by SDS-PAGE, andh) it catalyzes the transacylation of the γ-carboxyamide group inglutamine residue(s) in a peptide chain.
 4. The DNA of claim 3, whichhas at least the sequence of from the 118^(th) base to the 852^(nd) basein the base sequence of SEQ ID NO:2.
 5. A vector comprising a DNA codingfor a tranglutaminase obtained from Bacillus subtilis having thefollowing physicochemical properties:a) it is active between pH 7 andabout pH 9, b) it is active between about 40° C. and about 65° C., c) itis stable at about 60° C. or lower, d) the enzymatic activity of thetransglutaminase is not dependent on Ca⁺² and has an activity of 50% ormore in the presence of 5 mM of Ca⁺², e) it is inhibited byN-ethylmaleimide, cystamine or ammonium sulfate, f) it is not inhibitedby ethylenediamine-tetraacetic acid, dithiothreitol or2-mercaptoethanol, g) it has a molecular weight of (i) from about 18,000to about 22,000 as measured by gel permeation, and (ii) from about28,000 to about 30,000 as measured by SDS-PAGE, and h) it catalyzes thetransacylation of the γ-carboxyamide group in glutamine residue(s) in apeptide chain.
 6. A cell transformed with the vector of claim
 5. 7. Amethod for producing a transglutaminase, which comprises:a) incubating acell transformed with a vector comprising a DNA coding for atransglutaminase obtained from Bacillis subtilis in a culture medium tothereby produce and accumulate said transglutaminase in the medium or inthe cells, and b) collecting the transglutaminase, wherein thetransglutaminase has the following physicochemical properties:a) it isactive between pH 7 and about pH 9, b) it is active between about 40° C.and about 65° C., c) it is stable at about 60° C. or lower, d) theenzymatic activity of the transglutaminase is not dependent on Ca⁺² andhas an activity of 50% or more in the presence of 5 mM of Ca⁺², e) it isinhibited by N-ethylmaleimide, cystamine or ammonium sulfate, f) it isnot inhibited by ethylenediamine-tetraacetic acid, dithiothreitol or2-mercaptoethanol, g) it has a molecular weight of (i) from about 18,000to about 22,000 as measured by gel permeation, and (ii) from about28,000 to about 30,000 as measured by SDS-PAGE, and h) it catalyzes thetransacylation of the γ-carboxyamide group in glutamine residue(s) in apeptide chain.